Systematic Analysis of Cotton Non-specific Lipid Transfer Protein Family Revealed a Special Group That Is Involved in Fiber Elongation

Abstract

Non-specific lipid transfer proteins (nsLTPs) had been previously isolated from cotton fiber but their functions were unclear so far. Bioinformatic analysis of the tetraploid cotton genome database identified 138 nsLTP genes, falling into the 11 groups as reported previously. Different from Arabidopsis, cacao, and other crops, cotton type XI genes were considerably expanded and diverged earlier on chromosome At11, Dt11, and Dt08. Corresponding to the type XI genes, the type XI proteins (GhLtpXIs) all contained an extra N-terminal cap resulting in larger molecular weight. The research revealed that the expression of type XI genes was dramatically increased in fibers of tetraploid cotton compared with the two diploid progenitors. High-level of GhLtpXIs expression was observed in long-fibered cotton cultivars during fiber elongation. Ectopic expression of GhLtpXIs in Arabidopsis significantly enhanced trichome length, suggesting that GhLtpXIs promoted fiber elongation. Overall, the findings of this research provide insights into phenotypic evolution of Gossypium species and regulatory mechanism of nsLTPs during fiber development. HIGHLIGHT A specific group, type XI nsLTPs, was identified with predominant expression in elongating fibers of Gossypium hirsutum based on evolutionary, transcriptional, and functional analyses.

Keywords: cotton; expansion; fiber development; fiber evolution; nsLTP; type XI.

Figure 1

Multiple sequence alignment of GhLtp proteins. The gene ID and names of different types of GhLtps are presented with different colors. The conserved cysteine residues are marked against pink backgrounds. Consensus residues are marked by rectangles.

Figure 2

Phylogenetic tree of nsLtps in eight species. The full length of mature protein sequences of nsLTPs from G. hirsutum, G. arboreum, G. raimondii, Arabidopsis, B. rapa, Th. cacao, O. sativa, and V. vinifera were used to construct the phylogenetic tree using a Neighbor-Joining method. Lines of different colors represent classification of nsLTPs. Different species were marked at the end of the lines. Greek numerals present the corresponding type of genes that cannot be displayed by lines.

Figure 3

Distribution of GhLtps in 26 chromosomes. Chromosome numbers are indicated above each vertical bar. The scale represents centimorgan (cM). The segmental duplicated gene pairs are connected with red lines. The tandem duplicated gene clusters are marked in red perpendicular lines.

Figure 4

Differential expression analysis of GhLtps. (A) Volcano plot shows expression comparison between HY405 vs. CCRI8 and HY405 vs. ND601 represented by round and square points, respectively. FC stands for fold change. (B) Pie chart shows the proportion of gene types in differentially expressed GhLtps. (C) Heatmap shows expression of differentially expressed genes between cultivars with longer and shorter fibers. Data shown were log₂-transformed RPKM. Developmental stages of fiber are indicated in days post anthesis (DPA) above. The color bar represents the relative expression level. (D) Heatmap shows expression of differentially expressed GhLtps compared with their orthologs in G. raimondii or G. arboreum. Data shown were log₂-transformed FPKM of each gene which was quantified using RNA-seq data downloaded from CottonFGD. Developmental stages of fiber are indicated in days post anthesis (DPA) above. The color bar represents the relative expression level.

Figure 5

Temporal expression of nine GhLtpXIs in developing fibers. Ovules were collected on the day of anthesis and fibers were harvested on 5, 10, 15, and 20 DPA. Gene expression levels determined by qPCR were normalized to UBQ14 expression and shown as relative values to the maximal gene expression levels set at 100%. Error bars indicate SD of three biological replicates.

Figure 6

GhLtpXIs regulated trichome development in Arabidopsis. (A) Upregulation of GhLtpXIs in independent overexpression transgenic plants correlated to the length of trichomes on the surface of mature rosette leaves. Trichome length was measured under microscope and calculated for average of 30~50 trichomes. Asterisks indicate the significant difference in relation to WT (T-test, ^*p < 0.05, ^**p < 0.01). Expression of GhLtpXIs was determined by qPCR in leaves of each independent transgenic plants. Transcripts of GhLtpXIs were undetectable in WT. Results were normalized against the expression of TUB2. The maximum expression of each GhLtpXI is set as 100%. Error bars indicate SD. (B) Overexpressing of GhLtpXIs in transgenic plants promoted trichome length. Trichomes were observed under microscope after decolorization of leaves. The bar represents 100 μm.